The self-association of subunits of D-amino acid oxidase holoenzyme was studied by high-speed gel filtration with a short column of TSK-GEL G3000 SW in 0.1M sodium pyrophosphate (pH 8.3) at 25°C. Over the range of the peak concentrations of 0.009-4.45mg/ml in the presence of FAD the apparent Stokes radii increased with an increase of the concentrations and did not level off. The largest value obtained in this study was 61.5 Å. This would correspond to that calculated for the hexamer with linear subunit arrangement which has the largest Stokes radius among the various arrangements. These results provide the first gel chromatographic evidence that the higher polymers greater than the dimer participate in the self-associating system of the enzyme.